Preconditioning (PC) is a phenomenon where brief episodes of short periods of ischemia render the heart resistant to a sustained ischemic injury. In addition to PC, rapid ventricular pacing has also been shown to precondition the heart against ischemia/reperfusion (I/R) injury although the molecular mechanism of protection is currently unknown. Considering the potential clinical utility of rapid pacing in patients with coronary artery disease, a thorough investigation into the mechanisms of this form of PC is necessary. The central hypothesis of this proposal is that rapid ventricular pacing generates nitric oxide (NO), reactive oxygen species (ROS), and peroxynitrite (PN) which trigger downstream signaling events involving phosphorylation of MAP kinases, activation of nuclear factor KB (NFKB) and opening of mitochondrial KATP channel resulting in cardioprotection. Our first hypothesis is that rapid pacing induces early and delayed cardioprotection against myocardial infarction and apoptosis by initiating intracellular signaling events involving generation of NO, ROS and PN in the heart. Using antioxidants, PN decomposing catalysts, SOD transgenic mice and NOS knockout mice, we will establish direct role of ROS/NOS signaling in RVP induced cardioprotection. Our second hypothesis is that rapid pacing triggers signaling pathway leading to the phosphorylation of p38, p44/p42 (ERK I & 2) and p46/p54 JNK kinases. Using selective blockers we will determine the role of these kinase(s) in cardioprotection. The third hypothesis is that rapid ventricular pacing activates NFKB leading to the delayed cardioprotective effect. Using specific antisense oligonucleotides directed against the p65 subunit of NFKB, proteasome or nuclear localization inhibitors, overexpression of IkBa mutant with adenoviral constructs and p50 gene knockout mice we will determine the cause and effect of p50 and p65 subunits of NFKB in RVP-induced delayed cardioprotection. Our fourth hypothesis is that opening of mitochondrial KATP channel following rapid pacing is the common mediator of early and delayed protection against myocardial infarction. The proposed hypotheses will be examined using broad multidisciplinary approaches that will combine techniques including integrative physiology, molecular biology, genetically engineered mice and in vivo gene transfer. The information obtained from these studies may help us design novel therapies for protection of the heart against I/R injury.